The present invention relates to chimeric G proteins, expression constructs therefor, yeast cells expressing such constructs and methods of making and using them.
G protein-coupled receptors are integral membrane proteins, characteristically with 7 transmembrane domains, which convey hormonal and sensory signals to the cell interior (see (1) for review). These receptors are commonly referred to as 7TM receptors or 7TMRs. The receptors respond to ligand binding by activating heterotrimeric G proteins composed of xcex1, xcex2 and xcex3 subunits. The Gxcex1 subunit is bound to GDP in the G protein trimer, and interaction with an activated (ligand bound) receptor induces the replacement of GDP with GTP. Accompanying conformational changes result in the dissociation of Gxcex1-GTP and the Gxcex2/Gxcex3 particle, either of which can modulate ion channel or enzyme effectors to cause signal propagation. The signal persists until Gxcex1, which has GTPase activity, hydrolyses the bound GTP, allowing reassembly of the heterotrimer. Members of the RGS (Regulator of G protein Signalling) protein family regulate signal duration by acting as GAPs (GTPase activating proteins) for the Gxcex1 subunit (38) (17). The G protein signalling system appears to be common to all eukaryotes.
A well-characterised example of the trimeric G protein signalling system is the pheromone response pathway of the budding yeast Saccharomyces cerevisiae (20). Cells of the MATa mating type express a receptor encoded by the STE2 gene. This receptor becomes activated upon binding of the xcex1-factor mating pheromone, a peptide secreted by cells of the opposite (MATxcex1) mating type. The yeast G protein is assembled from the products of the GPA1 (Gxcex1), STE4 (Gxcex2), and STE18 (Gxcex3) genes. The Gxcex2/Gxcex3 (Ste4p/Ste18p) particle released upon activation of the Ste2p receptor conveys the signal to a mitogen-activated protein kinase (MAPK) module. This leads to activation of the cyclin-dependent kinase inhibitor Far1p, causing cell cycle arrest and transcriptional induction of a set of genes involved in the mating process, including FUS1. The pathway is desensitised by Sst2p, a member of the RGS family. Cells of the opposite mating type (MATxcex1) express a different receptor (Ste3p) and thereby respond to the pheromone (a-factor) secreted by MATa cells; otherwise the signalling apparatus utilised in the two mating types is the same.
At present, at least 16 Gxcex1 subunits, 5 Gxcex2 subunits and 11 Gxcex3 subunits have been identified in mammals, which can assemble a wide diversity of trimeric G proteins. On the basis of sequence homology, the Gxcex1 subunits fall into at least four families, related to Gxcex1i, Gxcex1s, Gxcex1q, or Gxcex112. Typically, a given 7TM receptor activates only a single or small subset of Gxcex1 subunits. Thus even in cells which express multiple Gxcex1 subunits, signalling may be specific to particular G proteins and their downstream signalling pathways. A combination of approaches has defined several specific regions of the Gxcex1 subunit as key determinants of receptor/G protein specificity. These include regions in the N-terminus, the switch II to switch III regions (21) which are primarily responsible for binding Gxcex2/Gxcex3, and particularly regions of the C-terminus. For example, a cluster of mutations occurring in the C-terminal region of Gxcex1 have been found to cause defects in receptor coupling ((29)(13) and references therein). Also, peptides modelled on the C-termini of Gxcex1t (transducin) and Gxcex1i2 compete for binding to rhodopsin and the xcex2-adrenergic receptor, respectively (9)(30)(35) and antibodies directed against the Gxcex1 C-terminus can also block interaction with receptors (6)(10)(36). The normal response to agonist stimulation of the adenosine A1 receptor in cultured mammalian cells (e.g. COS cells) is the activation of Gxcex1i-family proteins, resulting in inhibition of adenylate cyclase. Gxcex1q-containing heterotrimers in contrast do not normally respond to A1 activation. However, Gxcex1q subunit can be induced to interact with the A1 receptor by exchanging the C-terminal four amino acids of Gxcex1q for the corresponding amino acids of Gxcex1i2 (7). Hence, phospholipase C (an effector of activated Gxcex1q) can be activated by adenosine A1 receptor agonists when signalling is mediated by a chimeric Gxcex1 subunit (7). The somatostatin SST3 receptor is incompatible with Gxcex1s, but can be coupled to the activation of adenylate cyclase in COS cells by replacement of five C-terminal amino acids of Gxcex1s with corresponding residues from either Gxcex1i2, which is known to interact with the SST3 receptor, or from Gxcex116(18). Gxcex115 and Gxcex116 interact with a wide range of 7TM receptors (26), and are unusual in this respect. In crystal structures which have been solved for Gi1 (41) and Gt (19) trimers, the Gxcex1 C-terminal tail lies on a flat, largely uncharged surface of the G protein trimer which also includes two lipid groups covalently attached to the Gxcex1 N-terminus and the Gxcex3 C-terminus. This surface is believed to face the membrane and to be involved in interactions with the intracellular loops of the 7TM receptor.
Several reports have demonstrated that the yeast G protein signalling system can be coupled to heterologously expressed mammalian G protein-coupled (7TM) receptors. Some receptors, including the rat somatostatin SST2 receptor (33), and the rat adenosine A2areceptor (34), can interact directly with the yeast Gxcex1 protein Gpa1p, whereas other receptors, including the human growth hormone releasing hormone receptor (GHRHR)(12), are incompatible with Gpa1p. In order to allow coupling of these receptors, the yeast Gxcex1 subunit can be deleted and the heterologous receptor co-expressed with a full-length mammalian Gxcex1 subunit. Alternatively, chimeric Gxcex1 subunits have been used in which the C-terminal domain (approx. one third of the peptide sequence) of Gpa1p is replaced with the equivalent region of a mammalian Gxcex1 subunit. See WO95/21925 (American Cyanamid Company) for both approaches. Chimeras, or other modified or heterologous Gxcex1 subunits must satisfy several criteria to be useful in a yeast coupling system. Most importantly, they must bind efficiently to yeast Gxcex2/Gxcex3 to prevent signalling in the absence of activated 7TM receptor, and they must effectively interact with agonist-bound activated receptors to be capable of signal propagation.
Such heterologous or chimeric Gxcex1 components can facilitate the coupling of a heterologous 7TM receptor to the yeast signalling system (the pheromone response pathway) so that the effects of ligands such as potential new drug molecules on the receptor can be observed in phenotypic responses of the yeast cells. For example, cells may be induced to grow, and/or to convert an indicator dye compound in response to receptor agonists by introducing reporter genes downstream in the signalling pathway. However, chimeric constructs such as those described in WO95/21925 commonly show reduced affinity for the yeast G protein xcex2xcex3 subunit pair and show increased background signalling. Because of the specificity of a given receptor for one or a small number of the known Gxcex1 subunits, different Gxcex1 constructs have been required to demonstrate functional coupling activity with the majority of receptors tested.
The discovery of new drugs able to act via 7TM receptors requires screens of high efficiency, yet high specificity. Currently, the yeast coupling system is constrained by receptor/G protein specificity which imposes a requirement for a wide variety of Gxcex1 constructs to be tried for each receptor tested for coupling. For receptors which fail to couple, there is uncertainty as to whether failure is due to receptor/G protein incompatibility, or other reasons such as inappropriate receptor conformation, for example. It would be desirable to be able to couple a number of different receptors to the yeast G protein signalling pathway using a single Gxcex1 construct. This can be beneficial in the study of orphan receptors, of which nothing is known of receptor/G protein specificity. Alternatively, an array of Gxcex1 constructs could be used in the study of orphan receptors.
Surprisingly, we have found that certain chimeric G proteins, which we refer to as G protein xe2x80x9ctransplantsxe2x80x9d can be at least ten-fold more efficient than previously known chimeras in coupling mammalian 7TM receptors to the yeast mating pathway.
Accordingly, in a first aspect the present invention provides a chimeric Gxcex1 protein having yeast Gxcex1 (Gpa1p) amino acid sequences modified by a minimum of 3 amino acids positions within the C-terminal 10 amino acids by substitution with alternative amino acids. Preferably the chimera includes at least 5 such substitutions.
Accordingly, in a second aspect the present invention provides a chimeric Gxcex1 protein having yeast Gxcex1 (Gpa1p) amino acid sequences modified at a minimum of three amino acid positions within the C-terminal 10 amino acids by substitution with amino acids from a heterologous Gxcex1 protein. Preferably the chimera includes at least 5 substitutions. In preferred embodiments of this aspect of the invention, the chimera includes at least 3, desirably at least 5 consecutive amino acids corresponding to an amino acid sequence derived from the C-terminal 10 amino acids of a heterologous Gxcex1 protein. For example, the C-terminal 5 amino acids of a mammalian Gxcex1 protein, for example Gxcex116, but feasibly any Gxcex1, may replace at least the C-terminal 5 amino acids of the endogenous yeast Gxcex1 protein to provide a chimera according to the invention. We have designated such chimeras xe2x80x9ctransplantsxe2x80x9d or xe2x80x9ctransplant chimerasxe2x80x9d.
The chimeric Gxcex1 proteins of the present invention are believed to adopt a conformation which is more compatible with signal transduction in yeast (and is presumed to be closer to the native yeast Gxcex1 conformation) than that of previously known chimeras in which the C-terminal domain of the yeast protein was exchanged for that of a mammalian Gxcex1 protein. Thus the chimeras of the present invention have the advantage of changing receptor specificity and permitting the coupling of diverse receptors to the yeast signalling pathway with only minimal changes to the Gpa1p sequence. As a result of the close similarity to wild-type Gpa1p, these chimeric Gxcex1 subunits retain a similar affinity for Gxcex2/Gxcex3 as Gpa1p, and expression can be directed by the GPA1 promoter to achieve an optimal stoichiometry of Gxcex1. Expression from various other promoters is also compatible with receptor coupling.
In a third aspect, the present invention also provides a nucleotide sequence encoding a chimeric Gxcex1 protein according to the present invention. Also provided is an expression vector comprising said nucleotide sequence and capable of expressing the nucleotide sequence on transfection into a suitable host cell. The construction of expression vectors including suitable promoters, transcription termination sequences and marker genes will be apparent to a person skilled in the art. The host cell may desirably be a yeast cell of the species Saccharomyces cerevisiae and represents a further aspect of the invention.
Accordingly, the invention also provides a transformed yeast cell comprising a nucleotide sequence which encodes a chimeric Gxcex1 protein according to the present invention, for example a heterologous G protein-coupled receptor including receptors for which the ligand is unknown, and a nucleotide sequence which encodes a chimeric Gxcex1 protein comprising yeast Gxcex1 (Gpa1p) amino acid sequences and at least 3 amino acids derived from the amino acid sequence of the C-terminal 10 amino acids of a Gxcex1 protein according to the present invention for example a heterologous Gxcex1 protein, such as the mammalian, Gxcex116 protein. The heterologous receptor may be a 7TM receptor. Such receptors include those for acetylcholine, adrenaline, noradrenaline, dopamine, histamine, melatonin, serotonin, angiotensin, prostaglandins, cannabinoids, neuropeptide Y, substance P, opioids, glucagon, angiotensin, bradykinin, chemokines, thrombin, glycoprotein hormones, adenosine, nucleotides, and somatostatin.
Transformed yeast cells according to the present invention may also comprise a nucleotide sequence encoding a reporter gene operatively associated with a promoter responsive to the G protein signalling pathway. Such reporter genes may include HIS3 or other auxotrophic markers (such as URA3, LEU2, or TRP1) or genes which confer resistance or sensitivity to drug selections, such as CYH2 or G418R or other genes such as those encoding intracellular enzymes such as xcex2-galactosidase (LacZ) and luciferase, or green fluorescent protein (GFP), or genes encoding secreted enzymes such as a phosphatase such as PHO5, or a kinase. Desirably, yeast cells may contain combinations of multiple reporter genes, such as FUS1-HIS3 and FUS1-lacZ. In preferred embodiments the transformed yeast cells will also include mutations in at least one of the GPA1, SST2 or STE2 genes. Preferably such mutations will be deletions. Desirably, FAR1 is also deleted when a reporter gene is used to monitor activity in the G protein signalling pathway. This ensures that growth can continue even under conditions which activate the pheromone response pathway. In alternative embodiments of this invention, the FAR1 gene may remain intact so that agonist stimulation causing activation of the signalling pathway may be monitored as resulting in growth arrest.
Preferred yeast strains will have deletions of SST2 and GPA1 (yeast Gxcex1)xe2x80x94the former to prevent down-regulation of the signal by Sst2p activation of GTP-ase, the latter to prevent signal quenching, which occurs when Gxcex1 is present in stoichiometric excess to Gxcex2/Gxcex3, due to rapid reassociation of the actively signalling Gxcex2/Gxcex3 moiety into the inactive heterotrimer.
We have designated the chimera between the N-terminal 467 amino acids of Gpa1p and the 5 C-terminal amino acids of Gxcex116 as the Gpa1-Gxcex116 transplant. Similarly, the chimera containing the C-terminal 5 amino acids of Gxcex1q is designated as the Gpa1-Gxcex1q transplant, and the chimera containing the C-terminal 5 amino acids of Gxcex1s is designated as the Gpa1-Gxcex1s transplant. In addition, the following transplants have also been constructed. They are identical to those described above, ie. they comprise the N-terminal 467 amino acids of-Gpa1p and the C-terminal 5 amino acids from a Gxcex1 protein as follows: Gpa1-Gxcex112, Gpa1-Gxcex113, Gpa1-Gxcex114, Gpa1-Gxcex1i1, Gpa1-Gxcex1i3, Gpa1-Gxcex10 and Gpa1-Gxcex1z.
A further transplant was prepared between the yeast Gxcex1, Gpa1p and Gpa3, a Gxcex1 subunit from the yeast Ustilago maydis. This transplant is designated Gpa1-Gpa3. These transplants illustrate that by changing the 5 C-terminal amino acids of Gpa1p the specificity of the Gxcex1 subunit for receptors can be altered. Also the results indicate that the effectiveness of the xe2x80x9ctransplantsxe2x80x9d in coupling to foreign receptors in yeast is unexpectedly good compared to chimeric subunits with longer heterologous Gxcex1 regions.
In mammalian cells, the purinergic nucleotide P2Y2 receptor is coupled to the activation of phospholipase Cxcex2 (PLCxcex2) via Gxcex1q. We have found that the Gpa1-Gxcex1q transplant substantially improves the weak response to agonist observed with wild-type Gpa1p. Similarly, coupling of the somatostatin SST2 receptor achieved with the Gpa1-Gxcex116 transplant was enhanced ten-fold compared to either wild-type Gpa1p or the Gxcex1i/0 family chimeras, which is remarkable considering this receptor interacts with Gxcex1i/0 proteins in mammalian cells. Also the 5HT1A receptor can interact with the Gpa1-Gxcex116 transplant, even though it fails to stimulate wild-type Gpa1p in MMY9 yeast cells. Minimal amino acid substitutions can confer on yeast Gpa1p the properties of a generic G protein (Gxcex116) which was not possible under previously described approaches to chimera construction. Therefore this invention presents for the first time the possibility of a system comprising a single Gxcex1 subunit and able to couple a wide variety of 7TM receptors.
Moreover, we have found that the approach of substituting the five C-terminal amino acids of Gpa1p to generate the transplants is widely applicable, in that we have generated transplants of representative members of all four Gxcex1 families: Gxcex1i, Gxcex1s, Gxcex1q and Gxcex112. This was not possible in previously described approaches to chimera construction. Furthermore, all of the transplants can be expressed from the promoter of the GPA1 gene to achieve optimal stoichimetry for efficient coupling. This was not possible in previously described approaches to chimera construction, as some of these chimeras required expression from stronger promoters, as in the case of Gpa1/Gxcex1s (ref:12). Lastly, the pheromone response pathway is not activated in cells which express integrated versions of the transplants in the absence of activated receptors. This indicates that manipulations to the C-terminal amino acids do not interfere with the interaction with Gxcex2/Gxcex3; this was not true with previously described approaches to chimera construction. Together, our data suggest that the approach of creating transplants will be applicable to any newly discovered mammalian Gxcex1, or to Gxcex1 subunits not described herein (Gxcex1t or Gxcex1olf) or to Gxcex1 subunits derived from any other metazoan species.